Gd3+ and Mn2+ spin labelling

Electron-paramagnetic resonance (EPR) techniques, specifically double electron-electron resonance DEER (or PELDOR) are highly effective for determining the distances between two strategic sites in biological macromolecules such as proteins, nucleic acids, and their assemblies. These are usually carried out in frozen solutions and provide, for example, sparse structural information that can be used for tracking conformation changes upon ligand/substrate binding, can be used as constraints for modeling of unknown structures, and can determine how individual protein subunits with known structures assemble into larger structures. Such measurements are particularly needed for systems that present a challenge for X-ray crystallography or NMR analysis, such as membrane proteins in their natural environment. At the heart of this methodology lies the controlled labeling of the molecules of interest with paramagnetic probes, between which the distances are measured. These could be either intrinsic paramagnetic centers such as paramagnetic transition metal ions and radicals or artificially introduced spin labels.

The field of spin labeling has been dominated by nitroxide stable radicals since it was first introduced by McConnell in the 1960s. Although highly polular, at conventional EPR frequencies DEER sensitivity limitations leave many important probelms outside its applicability range. In addition nitroxides have limited stability within living cells.

Membrane proteins present a challenge also for DEER measurements. This is because the localization of the spin labeled proteins in the membrane bilayer results in high local concentration that reduces considerably the phase memory time. This, in turn reduces sensitivity and makes long distances hard to access.

The above-mentioned limitations prompted my lab, together with our collaborators, to introduce new spin labels for DEER measurements at Q- and W-band frequencies. These are based on Gd3+, which is a half integer, high-spin ion (S=7/2) with half-filled valence f orbitals that exhibit high EPR sensitivity at high magnetic fields. In addition, Gd3+ chelates and their derivatives are routinely used as contrast agents in MRI. As opposed to nitroxides, they are stable on the cell; this is most important in terms of future development of in-cell DEER.

Since our first demonstration of Gd3+ - Gd3+ DEER on a model compound in 2007, there have been a number such distance measurements on model systems, peptides, proteins, nucleic acids, and coated nanoparticles. Recently we also demonstrated the potential of W-band Gd3+-Gd3+ DEER distance measurements in a membrane environment using trans-membrane model peptides. The results featured high absolute sensitivity (>0.15 nmols). We are currently applying this new methodology to study protein functions, examples are protein conformational changes upon ligand binding, peptide membrane interaction and protein oligomerization.

Mn2+, being also a half integer high spin ion, has spectroscopic properties similar to Gd3+ and currently we are exploring its potential as a spin label as well. Finally the introduction of Gd3+ and Mn2+-spin labels allow to combined different types of labels in distance measurements, such as Gd3+ nitroxide  and Mn2+-nitroxide.



  • Goldfarb D., Gd3+ spin labeling for distance measurements by pulse EPR spectroscopy. Physical Chemistry Chemical Physics 2014, 16: p. 9685-9699.
  • Feintuch, A.; Otting, G.; Goldfarb, D. Gd3+ Spin Labeling for Measuring Distances in Biomacromolecules: Why and How? Methods in Enzymology 2015, 563, 415–457.
  • Raitsimring, A. M., Gunanathan, C., Potapov, A., Efremenko, I., Martin, J. M., Milstein, D., Goldfarb, D., Gd3+ complexes as potential spin labels for high field pulsed EPR distance measurements, Journal of the American Chemical Society 2007, 129, 14138-9
  • Potapov, A., Yagi, H., Huber, T., Jergic, S., Dixon, N.E., Otting, G.Goldfarb, D. Nanometer-scale distance measurements in proteins using Gd3+ spin labeling. Journal of the American Chemical Society 2010, 132,9040-8
  • Gordon-Grossman, M.; Kaminker, I.; Gofman, Y.; Shai, Y.; Goldfarb, D., W-Band pulse EPR distance measurements in peptides using Gd3+–dipicolinic acid derivatives as spin labels, Physical Chemistry Chemical Physics 2011, 13, 10771
  • Matalon, E.; Huber, T.; Hagelueken, G.; Graham, B.; Frydman, V.; Feintuch, A.; Otting, G.; Goldfarb, D., Gadolinium(III) Spin Labels for High-Sensitivity Distance Measurements in Transmembrane Helices, Angew. Chem. Int. Ed. 2013, 52, 11831
  • Yagi, H.; Banerjee, D.; Graham, B.; Huber, T.; Goldfarb, D.; Otting, G., Gadolinium Tagging for High-Precision Measurements of 6 nm Distances in Protein Assemblies by EPR, J. Am. Chem. Soc. 2011, 133, 10418.